Project Summary/Abstract In the endocytic system, vesicles from the plasma membrane containing lipids and integral membrane proteins fuse with sorting endosomes. Lipids and proteins are sorted there and delivered back to the plasma membrane or to other organelles by the process of retrograde trafficking. The retromer complex is involved in multiple retrograde pathways and is a major driver of retrograde trafficking. Retrograde trafficking is essential for the normal development and maintenance of cells and tissues. Defects in retrograde trafficking can result in dysregulation and disease, and have been implicated in disorders like Alzheimer's, Parkinson's, diabetes, osteoporosis, and retinal degeneration. The goal of this project is to determine how retromer sorts cargo and packages it into the sorting/carrier tubules of the endosome, from which endosomal carrier vesicles will subsequently be separated. Aim 1 will define how integral membrane proteins are captured and organized for export by retromer. Aim 2 will determine the mechanism by which cargo that has been captured by retromer is packaged into carrier tubules. These aims will be accomplished by examination of sorting dynamics in a reconstituted endosomal system. Protein components of the endosomal sorting system (membrane protein cargo, retromer complex, Rab7, SNX3, Fam21, WASH complex, Arp2/3 complex, actin, and SNX-BAR) will be reconstituted into supported bilayers (SBLs) and giant unilamellar vesicles (GUVs). The reconstitution system will be used to test mechanistic hypotheses about how retromer sorts and packages cargo. The findings will be corroborated by parallel studies in live cells.